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Introduction and hypothesisWe aimed to analyze the quality of sexual life of patients with apical vaginal wall prolapse who had undergone laparoscopic lateral suspension (LLS) and laparoscopic sacrocolpopexy (LSC).MethodsWe performed a secondary analysis of sexual outcomes of a previous randomized control trial comparing LLS and LSC in 89 women with symptomatic POP stage ≥ II. We evaluated sexually active (SA) and non-sexually active women (NSA) using the Pelvic Organ Prolapse/Incontinence Sexual Questionnaire-IUGA-Revised (PISQ-IR). Women were reviewed over a period of 1 year post-surgery.ResultsAnalysis of the entire PISQ-IR questionnaire indicates that surgical treatment of POP resulted in an improvement of the quality of sexual life in 21 (80.76%) in the group of sexually active women after LSC and in 20 (83.33%) in the group of SA patients after LLS. In both groups of patients, dyspareunia was not observed.ConclusionIn conclusion, the quality of sexual life in SA group of patients improved significantly after both surgical procedures. The quality of sexual life of surveyed women significantly improved after curing POP symptoms.

IntroductionThe COVID-19 pandemic caused by the SARS-CoV-2 virus has underscored the urgent necessity for the development of antiviral compounds that can effectively target coronaviruses. In this study, we present the first evidence of the antiviral efficacy of hyperforin, a major metabolite of St. John’s wort, for which safety and bioavailability in humans have already been established.MethodsAntiviral assays were conducted in cell culture with four human coronaviruses: three of high virulence, SARS-CoV-2, SARS-CoV, and MERS-CoV, and one causing mild symptoms, HCoV-229E. The antiviral activity was also evaluated in human primary airway epithelial cells. To ascertain the viral step inhibited by hyperforin, time-of-addition assays were conducted. Subsequently, a combination assay of hyperforin with remdesivir was performed.ResultsThe results demonstrated that hyperforin exhibited notable antiviral activity against the four tested human coronaviruses, with IC50 values spanning from 0.24 to 2.55 µM. Kinetic studies indicated that the observed activity occur at a post-entry step, potentially during replication. The antiviral efficacy of hyperforin was additionally corroborated in human primary airway epithelial cells. The results demonstrated a reduction in both intracellular and extracellular SARS-CoV-2 viral RNA, confirming that hyperforin targeted the replication step. Finally, an additive antiviral effect on SARS-CoV-2 was observed when hyperforin was combined with remdesivir.DiscussionIn conclusion, hyperforin has been identified as a novel pan-coronavirus inhibitor with activity in human primary airway epithelial cells, a preclinical model for coronaviruses. These findings collectively suggest that hyperforin has potential as a candidate antiviral agent against current and future human coronaviruses.

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Abstract Background Despite recent advancements, limitations in the treatment and control of hepatitis C virus (HCV) infection reprioritized the studies for invention of an efficient HCV vaccine to elicit strong neutralizing antibodies (NAbs) and cellular responses. Methods Herein, we report molecular construction of a BacMam virus-based surface display for a subtype-1a HCV gpE2 (Bac-CMV-E2-gp64; Bac) that both expressed and displayed gpE2 in mammalian cells and bacouloviral envelope, respectively. Results Assessments by western blotting, Immunofluorescence and Immunogold-electron microscopy indicated the proper expression and incorporation in insect cell and baculovirus envelope, respectively. Mice immunized in three different prime-boost immunization groups of: Bac/Bac, Bac/Pro (bacoulovirus-derived gpE2) and Bac/DNA (plasmid DNA (pCDNA)-encoding gpE2) developed high levels of IgG and IFN-γ (highest for Bac/Bac group) indicating the induction of both humeral and cellular immune responses. Calculation of the IgG2a/IgG1 and IFN-γ/IL-4 ratios indicated a Th1 polarization of immune responses in the Bac/Bac and Bac/DNA groups but a balanced Th1-Th2 phenotype in the Bac/Pro group. Sera of the mice in the Bac/Bac group provided the highest percentage of cross-NAbs against a subtype-2a HCVcc (JFH1) compared to Bac/Pro and Bac/DNA groups (62% versus 41% and 6%). Conclusions Results indicated that BacMam virus-based surface display for gpE2 might act as both subunit and DNA vaccine and offers a promising strategy for development of HCV vaccine for concurrent induction of strong humoral and cellular immune responses.

Mounting evidence suggests that the gut-to-lung axis is critical during respiratory viral infections. We herein hypothesized that disruption of gut homeostasis during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may associate with early disease outcomes. To address this question, we took advantage of the Syrian hamster model. Our data confirmed that this model recapitulates some hallmark features of the human disease in the lungs. We further showed that SARS-CoV-2 infection associated with mild intestinal inflammation, relative alteration in intestinal barrier property and liver inflammation and altered lipid metabolism. These changes occurred concomitantly with an alteration of the gut microbiota composition over the course of infection, notably characterized by a higher relative abundance of deleterious bacterial taxa such as Enterobacteriaceae and Desulfovibrionaceae. Conversely, several members of the Ruminococcaceae and Lachnospiraceae families, including bacteria known to produce the fermentative products short-chain fatty acids (SCFAs), had a reduced relative proportion compared to non-infected controls. Accordingly, infection led to a transient decrease in systemic SCFA amounts. SCFA supplementation during infection had no effect on clinical and inflammatory parameters. Lastly, a strong correlation between some gut microbiota taxa and clinical and inflammation indices of SARS-CoV-2 infection severity was evidenced. Collectively, alteration of the gut microbiota correlates with disease severity in hamsters making this experimental model valuable for the design of interventional, gut microbiota-targeted, approaches for the control of COVID-19. Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019; SCFAs, short-chain fatty acids; dpi, day post-infection; RT-PCR, reverse transcription polymerase chain reaction; IL, interleukin. ACE2, angiotensin converting enzyme 2; TMPRSS2, transmembrane serine protease 2.

Background & Aims: Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker. Methods: The anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights. Results: AvFc was capable of neutralizing cell culture–derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed. Conclusions: These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing three open reading frames (ORFs). ORF1 encodes the ORF1 non–structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all three systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope ®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.

Millions of people are still infected with hepatitis C virus (HCV) nowadays. Although recent antivirals targeting HCV proteins are very efficient, they are not affordable for many people infected with this virus. Therefore, new and more accessible treatments are needed. Several Ivorian medicinal plants are traditionally used to treat “yellow malaria”, a nosological category including illness characterized by symptomatic jaundice such as hepatitis. Therefore, some of these plants might be active against HCV. An ethnobotanical survey in Côte d’Ivoire allowed us to select such medicinal plants. Those were first extracted with methanol and tested for their anti-HCV activity. The most active ones were further studied to specify their IC50 and to evaluate their toxicity in vitro. Greener solvents were tested to obtain extracts with similar activities. Following a phytochemical screening, tannins of the most active plants were removed before re-testing on HCV. Some of these tannins were identified by UPLC-MS and pure molecules were tested against HCV. Out of the fifteen Ivorian medicinal plants selected for their putative antiviral activities, Carapa procera DC. and Pericopsis laxiflora (Benth. ex Baker) Meeuwen were the most active against HCV (IC50: 0.71 and 0.23 μg/ml respectively) and not toxic for hepatic cells. Their crude extracts were rich in polyphenols, including tannins such as procyanidins A2 which is active against HCV. The same extracts without tannin lost their anti-HCV activity. Replacing methanol by hydro-ethanolic solvent led to tannins-rich extracts with similar antiviral activities, and higher than that of aqueous extracts.

Summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly rampaged worldwide, causing a pandemic of coronavirus disease (COVID -19), but the biology of SARS-CoV-2 remains under investigation. We demonstrate that both SARS-CoV-2 spike protein and human coronavirus 229E (hCoV-229E) or its purified S protein, one of the main viruses responsible for the common cold, induce the transient opening of Pannexin-1 (Panx-1) channels in human lung epithelial cells. However, the Panx-1 channel opening induced by SARS-CoV-2 is greater and more prolonged than hCoV-229E/S protein, resulting in an enhanced ATP, PGE2, and IL-1β release. Analysis of lung lavages and tissues indicate that Panx-1 mRNA expression is associated with increased ATP, PGE2, and IL-1β levels. Panx-1 channel opening induced by SARS-CoV-2 spike protein is angiotensin-converting enzyme 2 (ACE-2), endocytosis, and furin dependent. Overall, we demonstrated that Panx-1 channel is a critical contributor to SARS-CoV-2 infection and should be considered as an alternative therapy.

Study Objective: Evaluate the effects of a fast-track (FT) protocol on costs and post-operative recovery.Methods: One hundred and seventy women undergoing total laparoscopic hysterectomy for a benign indication were randomized in a FT protocol or a usual care protocol. A FT protocol included the combination of minimally invasive surgery, analgesia optimization, early oral refeeding and rapid mobilization of patients was compared to a usual care protocol. Primary outcome was costs. Secondary outcomes were length of stay, post-operative morbidity and patient satisfaction.Main Results: The mean total cost in the FT group was 13,070 ± 4,321 Euros (EUR) per patient, and that in the usual care group was 3.5% higher at 13,527 ± 3,925 EUR (p = 0.49). The FT group had lower inpatient surgical costs but higher total ambulatory costs during the first post-operative month. The mean hospital stay in the FT group was 52.7 ± 26.8 h, and that in the usual care group was 20% higher at 65.8 ± 33.7 h (p = 0.006). Morbidity during the first post-operative month was not significantly different between the two groups. On their day of discharge, the proportion of patients satisfied with pain management was similar in both groups [83% in FT and 78% in the usual care group (p = 0.57)]. Satisfaction with medical follow-up 1 month after surgery was also similar [91% in FT and 88% in the usual care group (p = 0.69)].Conclusion: Implementation of a FT protocol in laparoscopic hysterectomy for benign indications has minimal non-significant effects on costs but significantly reduces hospital stay without increasing post-operative morbidity nor decreasing patient satisfaction.Clinical Trial Registration:www.ClinicalTrials.gov, identifier: NCT04839263.

Abstract Background Although DAAs hold promise to significantly reduce rates of chronic HCV infections, its eradication still requires development of an effective vaccine. Prolonged T cell responses and cross neutralizing antibodies are ideal for vaccination against the infection. We aimed to design and synthesize a 6 multi epitope peptide vaccine candidate and provide evidence for production of extended cellular and neutralizing Abs in mice. Methods Six peptides derived from conserved epitopes in E1, E2 (n = 2),NS4B, NS5A and NS5B were designed, synthesized in a multiple antigenic peptide (MAP) form and administered w/o adjuvant to BALB/c mice as HCVp6-MAP at doses ranging from 800 ng to 16 μg. Humoral responses to structural epitopes were assayed by ELISA at different times after injection. ELISpot assay was used to evaluate IFN ɣ producing CD4+/ CD8+ T- lymphocytes at extended durations i.e. > 20 weeks. Viral neutralization by mice sera was tested for genotypes 2a (JFH1) and a chimeric 2a/4a virus (ED43/JFH1) in HCVcc culture. Results HCVp6-MAP confers potent viral neutralization and specific cellular responses at > 1600 ng/ animal for at least 20 weeks. Conclusion We report on a promising anti HCV vaccine for future studies on permissive hosts and in clinical trials.

Introduction: Ectopic pregnancy in a non-communicating rudimentary uterine horn is a rare gynecological condition associated with a high risk of uterine rupture and important maternal mortality and morbidity. A surgical excision of the rudimentary horn is the standard treatment, usually performed by laparotomy in the second trimester.Methods: A 36-year-old woman, secundigravida and nulliparous, was admitted to the emergency obstetric unit with acute pelvic pain. The ultrasound found an ectopic pregnancy at 15 weeks gestational age with fetal cardiac activity. As her hemodynamic status was stable, a diagnostic laparoscopy was performed and confirmed the development of the pregnancy in a left rudimentary uterine horn.Results: We report a total laparoscopic removal of a pre-ruptured rudimentary uterine horn containing a second trimester ectopic pregnancy, using a vessel-sealer device. To our knowledge, only three other cases of successful laparoscopic treatment of second trimester rudimentary horn pregnancies have been reported in the literature.Conclusion: Laparoscopy is an efficient and safe surgical option for treating rudimentary horn second trimester pregnancy in patients with hemodynamic stability.

Since December 2019, SARS-CoV-2 has spread quickly worldwide, leading to more than 280 million confirmed cases, including over 5,000,000 deaths. Interestingly, coronaviruses were found to subvert and hijack autophagic process to allow their viral replication. Autophagy-modulating compounds thus rapidly emerged as an attractive strategy to fight SARS-CoV-2 infection, including the well-known chloroquine (CQ). Here, we investigated the antiviral activity and associated mechanism of GNS561/Ezurpimtrostat, a small lysosomotropic molecule inhibitor of late-stage autophagy. Interestingly, GNS561 exhibited antiviral activity of 6–40 nM depending on the viral strain considered, currently positioning it as the most powerful molecule investigated in SARS-CoV-2 infection. We then showed that GNS561 was located in lysosome-associated-membrane-protein-2-positive (LAMP2-positive) lysosomes, together with SARS-CoV-2. Moreover, GNS561 increased LC3-II spot size and caused the accumulation of autophagic vacuoles and the presence of multilamellar bodies, suggesting that GNS561 disrupted the autophagy mechanism. To confirm our findings, we used the K18-hACE2 mouse model and highlighted that GNS561 treatment led to a decline in SARS-CoV-2 virions in the lungs associated with a disruption of the autophagy pathway. Overall, our study highlights GNS561 as a powerful drug in the treatment of SARS-CoV-2 infection and supports the hypothesis that autophagy blockers could be an alternative strategy for COVID-19.

The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.

Objective: The objective of this study was to review the records of patients with excised abdominal wall endometriosis (AWE) to determine patient characteristics, diagnostic methods, presence of concurrent pelvic endometriosis and type of surgery. Study design: Medical records from an 11-year period were searched to identify histologically confirmed AWE cases. Descriptive data were collected and analyzed. Two subgroups were differentiated: isolated AWE and pelvic endometriosis-associated AWE. Results: Thirty-five women with AWE were included. The most common symptom was cyclic abdominal or parietal pain (68.6%); 17.1% of the women had no symptoms. Twenty-nine women (82.8%) had a history of gynecological or obstetrical surgery, most commonly cesarean section (CS). The mean interval between prior surgery and appearance of symptoms was 5.3 years. Six women (17.1%) had no prior surgery; all six presented with umbilical nodules, nulliparity and confirmed mild to severe pelvic endometriosis. Among all patients, 34.3% had concurrent pelvic endometriosis, 40% presented with isolated AWE and 25.7% had no pelvic exploration. Women with concurrent pelvic endometriosis had significantly lower parity, smaller nodule size and a higher likelihood of umbilical location than those with isolated AWE (p

Vaccination remains one of the most effective tools to prevent infectious diseases. To ensure that the best possible antigenic components are chosen to stimulate a cognitive immune response, boosting antigen presentation using adjuvants is common practice. Nanodiamond-based adjuvants are proposed here as a rapid and versatile platform for antigen conjugation, utilizing peptides common to different pathogenic strains and making this strategy a good candidate for a “ready-to-use” vaccine. Initiation of an inflammatory reaction with a resulting immune response is based on the ability of living organisms to entrap nanostructures such as nanodiamonds with neutrophil extracellular traps (NETs) formation. In this work, coronavirus peptide homological for MERS-CoV, fusion inhibitor, was conjugated to nanodiamonds and used to induce neutrophilic-driven self-limiting inflammation. The resulting adjuvant was safe and did not induce any tissue damage at the site of injection. Mice immunization resulted in IgG titers of ¼,000 within 28 days. Immunization of rabbits resulted in the formation of a high level of antibodies persistently present for up to 120 days after the first immunization (animal lifespan ~3 years). The peptide used for immunization proved to be reactive with sera of convalescent COVID patients, demonstrating the possibility of developing pancoronaviral vaccine candidates.

The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), which is a protein involved both in genome replication and encapsidation. An NS5A-eGFP (enhanced green fluorescent protein) tagged version of the strain JFH-1-derived wild-type HCV was compared to the corresponding assembly-deficient viruses Δcore, NS5A basic cluster 352–533 mutant (BCM), and serine cluster 451 + 454 + 457 mutant (SC). These analyses highlighted an increase of NS5A motility when the viral protein core was lacking. Although to a lesser extent, NS5A motility was also increased in the BCM virus, which is characterized by a lack of interaction of NS5A with the viral RNA, impairing HCV genome encapsidation. This observation suggests that the more static NS5A population is mainly involved in viral assembly rather than in RNA replication. Finally, NS4B exhibited a reduced co-localization with NS5A and lipid droplets for both Δcore and SC mutants, which is characterized by the absence of interaction of NS5A with core. This observation strongly suggests that NS5A is involved in targeting NS4B to lipid droplets (LDs). In summary, this work contributes to a better understanding of the interplay between HCV proteins during the viral life cycle.

An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in the coronavirus disease pandemic, drastically affecting global health and economy. Though the understanding of the disease has improved, fighting the virus remains challenging. One of the strategies is repurposing existing drugs as inhibitors of SARS-CoV-2. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, reportedly inhibits the replication of RNA viruses, especially Coxsackieviruses B (CVB), such as CV-B4 in vitro and in vivo. Therefore, in this study, we investigated the in vitro antiviral activity of FLX against SARS-CoV-2 in a model of acute infection. When 10 μM of FLX was added to SARS-CoV-2-infected Vero E6 cells, the virus-induced cytopathic effect was not observed. In this model, the level of infectious particles in the supernatant was lower than that in controls. The level was below the limit of detection of the assay up to day 3 post-infection when FLX was administered before viral inoculation or simultaneously followed by daily inoculation. In conclusion, FLX can inhibit SARS-CoV-2 in vitro. Further studies are needed to investigate the potential value of FLX to combat SARS-CoV-2 infections, treat SARS-CoV-2-induced diseases, and explain the antiviral mechanism of this molecule to pave way for novel treatment strategies.

[This corrects the article DOI: 10.1371/journal.pone.0070809.].

Hepatitis C virus (HCV) envelope glycoprotein heterodimer, E1E2, plays an essential role in virus entry and assembly. Furthermore, due to their exposure at the surface of the virion, these proteins are the major targets of anti-HCV neutralizing antibodies. Their ectodomain are heavily glycosylated with up to 5 sites on E1 and up to 11 sites on E2 modified by N-linked glycans. Thus, one-third of the molecular mass of E1E2 heterodimer corresponds to glycans. Despite the high sequence variability of E1 and E2, N-glycosylation sites of these proteins are generally conserved among the seven major HCV genotypes. N-glycans have been shown to be involved in E1E2 folding and modulate different functions of the envelope glycoproteins. Indeed, site-directed mutagenesis studies have shown that specific glycans are needed for virion assembly and infectivity. They can notably affect envelope protein entry functions by modulating their affinity for HCV receptors and their fusion activity. Importantly, glycans have also been shown to play a key role in immune evasion by masking antigenic sites targeted by neutralizing antibodies. It is well known that the high mutational rate of HCV polymerase facilitates the appearance of neutralization resistant mutants, and occurrence of mutations leading to glycan shifting is one of the mechanisms used by this virus to escape host humoral immune response. As a consequence of the importance of the glycan shield for HCV immune evasion, the deletion of N-glycans also leads to an increase in E1E2 immunogenicity and can induce a more potent antibody response against HCV.

Bats are a reservoir for a diverse range of viruses, including coronaviruses (CoVs). To determine the presence of CoVs in French bats, fecal samples were collected between July and August of 2014 from four bat species in seven different locations around the city of Bourges in France. We present for the first time the presence of alpha-CoVs in French Pipistrellus pipistrellus bat species with an estimated prevalence of 4.2%. Based on the analysis of a fragment of the RNA-dependent RNA polymerase (RdRp) gene, phylogenetic analyses show that alpha-CoVs sequences detected in French bats are closely related to other European bat alpha-CoVs. Phylogeographic analyses of RdRp sequences show that several CoVs strains circulate in European bats: (i) old strains detected that have probably diverged a long time ago and are detected in different bat subspecies; (ii) strains detected in Myotis and Pipistrellus bat species that have more recently diverged. Our findings support previous observations describing the complexity of the detected CoVs in bats worldwide.

Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2. Their structure and mode of fusion remain unknown, and so does the virion architecture. The organization of the HCV envelope shell in particular is subject to discussion as it incorporates or associates with host-derived lipoproteins, to an extent that the biophysical properties of the virion resemble more very-low-density lipoproteins than of any virus known so far. The recent development of novel cell culture systems for HCV has provided new insights on the assembly of this atypical viral particle. Hence, the extensive E1E2 characterization accomplished for the last two decades in heterologous expression systems can now be brought into the context of a productive HCV infection. This review describes the biogenesis and maturation of HCV envelope glycoproteins, as well as the interplay between viral and host factors required for their incorporation in the viral envelope, in a way that allows efficient entry into target cells and evasion of the host immune response.

Hepatitis C Virus (HCV) assembly process is the least understood step in the virus life cycle. The functional data revealed by forward and reverse genetics indicated that both structural and non-structural proteins are involved in the assembly process. Using confocal and electron microscopy different groups determined the subcellular localization of different viral proteins and they identified the lipid droplets (LDs) as the potential viral assembly site. Here, we aim to review the mechanisms that govern the viral proteins recruitment to LDs and discuss the current model of HCV assembly process. Based on previous examples, this review will also discuss advanced imaging techniques as potential means to extend our present knowledge of HCV assembly process.

Despite the induction of effective immune responses, 80% of hepatitis C virus (HCV)-infected individuals progress from acute to chronic hepatitis. In contrast to the cellular immune response, the role of the humoral immune response in HCV clearance is still subject to debate. Indeed, HCV escapes neutralizing antibodies in chronically infected patients and reinfection has been described in human and chimpanzee. Studies of antibody-mediated HCV neutralization have long been hampered by the lack of cell-culture-derived virus and the absence of a small animal model. However, the development of surrogate models and recent progress in HCV propagation in vitro now enable robust neutralization assays to be performed. These advances are beginning to shed some light on the mechanisms of HCV neutralization. This review summarizes the current state of knowledge of the viral targets of anti-HCV-neutralizing antibodies and the mechanisms that enable HCV to evade the humoral immune response. The recent description of the HCV glycan shield that reduces the immunogenicity of envelope proteins and masks conserved neutralizing epitopes at their surface constitutes the major focus of this review.

Chronic Hepatitis C infection is a global health problem. While primary infection is often inapparent, it becomes chronic in most cases. Chronic infection with Hepatitis C virus (HCV) frequently leads to liver cirrhosis or liver cancer. Consequently, HCV infection is one of the leading causes for liver transplantation in industrialized countries. Current treatment is not HCV specific and is only effective in about half of the infected patients. This situation underlines the need for new antivirals against HCV. To develop new and more efficient drugs, it is essential to specifically target the different steps of the viral life cycle. Of those steps, the targeting of HCV assembly has the potential to abolish virus production. This review summarizes the advances in our understanding of HCV particle assembly and the identification of new antiviral targets of potential interest in this late step of the HCV life cycle.

More than 170 million patients worldwide are chronically infected with hepatitis C virus (HCV). Prevalence rates range from 0.5% in Northern European countries to 28% in some areas of Egypt. HCV is hepatotropic, and in many countries chronic hepatitis C is a leading cause of liver disease including fibrosis, cirrhosis and hepatocellular carcinoma. HCV persists in 50–85% of infected patients, and once chronic infection is established, spontaneous clearance is rare. HCV is a member of the Flaviviridae family, in which it forms its own genus. Many lines of evidence suggest that the HCV life cycle displays many differences to that of other Flaviviridae family members. Some of these differences may be due to the close interaction of HCV with its host’s lipid and particular triglyceride metabolism in the liver, which may explain why the virus can be found in association with lipoproteins in serum of infected patients. This review focuses on the molecular events underlying the HCV cell entry process and the respective roles of cellular co-factors that have been implied in these events. These include, among others, the lipoprotein receptors low density lipoprotein receptor and scavenger receptor BI, the tight junction factors occludin and claudin-1 as well as the tetraspanin CD81. We discuss the roles of these cellular factors in HCV cell entry and how association of HCV with lipoproteins may modulate the cell entry process.

The clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2's interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421-645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50's ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

Significant progress has been made in Hepatitis C virus (HCV) culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells' permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B) and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates.

Despite the introduction of effective treatments for hepatitis C in clinics, issues remain regarding the liver disease induced by chronic hepatitis C virus (HCV) infection. HCV is known to disturb the metabolism of infected cells, especially lipid metabolism and redox balance, but the mechanisms leading to HCV-induced pathogenesis are still poorly understood. In an APEX2-based proximity biotinylation screen, we identified ACBD5, a peroxisome membrane protein, as located in the vicinity of HCV replication complexes. Confocal microscopy confirmed the relocation of peroxisomes near HCV replication complexes and indicated that their morphology and number are altered in approximately 30% of infected Huh-7 cells. Peroxisomes are small versatile organelles involved among other functions in lipid metabolism and ROS regulation. To determine their importance in the HCV life cycle, we generated Huh-7 cells devoid of peroxisomes by inactivating the PEX5 and PEX3 genes using CRISPR/Cas9 and found that the absence of peroxisomes had no impact on replication kinetics or infectious titers of HCV strains JFH1 and DBN3a. The impact of HCV on peroxisomal functions was assessed using sub-genomic replicons. An increase of ROS was measured in peroxisomes of replicon-containing cells, correlated with a significant decrease of catalase activity with the DBN3a strain. In contrast, HCV replication had little to no impact on cytoplasmic and mitochondrial ROS, suggesting that the redox balance of peroxisomes is specifically impaired in cells replicating HCV. Our study provides evidence that peroxisome function and morphology are altered in HCV-infected cells.

Introduction Transvaginal natural orifice transluminal endoscopic surgery (vNOTES) has already proven its non-inferiority to conventional abdominal laparoscopy (CAL) for hysterectomy without conversion. The results in terms of efficacy and safety are promising. However, we note a lack of medical literature and no specific randomised controlled trial assessing women’s sexual function after vNOTES for benign adnexal surgery. The aim of this RCT is to confirm the non-inferiority of the vNOTES approach for benign adnexal pathology compared with CAL on women’s sexual function. Secondary outcomes will evaluate vNOTES’s efficiency, morbidity and postoperative complications compared with CAL for benign adnexal surgery. The relationship between adnexal mass morcellation and the quality of the histological analysis will also be evaluated as secondary outcome.Methods and analysis Women aged 18–70 years undergoing a benign adnexal surgery at the Geneva University Hospitals will be eligible and randomised with a 1:1 ratio to the CAL arm or the vNOTES arm, if inclusion criteria are met. Participants will complete the Female Sexual Function Index, the Couple Satisfaction Index-16 and a self-reported questionnaire on dyspareunia within 4 weeks prior to randomisation and at 3+6 months after surgery. General and clinical data will be collected when the patient is enrolled in the study, during hospitalisation and at 1 month postoperative to assess secondary outcomes.An absence of impairment on sexual function will be confirmed with a stability or an improvement of the evaluated scores in each group at 3 and 6 months postoperative compared with the preoperative scores. We expect to have no statistically significant difference in sexuality questionnaires scores between the two groups.Ethics and dissemination Protocol of this study was validated by the Cantonal Research Ethics Commission of Geneva, Switzerland, on 9 August 2022. We aim to publish the study’s results in peer-reviewed journals within 3 years.Trial registration number NCT05761275.

Uterine leiomyoma is the most common benign tumour of the uterus in women of reproductive age. When removed surgically, a mini-invasive procedure is preferentially used (laparoscopic or robotic) and the extraction of the specimen can be managed by power morcellation. In this consecutive case-series, we present three cases of parasitic leiomyoma that appeared following previous surgical management of leiomyoma using the technique of laparoscopic myomectomy with uncontained power morcellation. The time frame in between the initial surgery and the diagnosis of the parasitic leiomyoma was 5.7 years. All three patients were diagnosed with endometriosis: 2 cases prior to the initial surgery and 1 case after the initial surgery. One hypothesis could be that, due to pelvic inflammation, endometriosis is a risk factor for iatrogenic parasitic leiomyoma development in case of uncontained morcellation of leiomyoma during myomectomy.

The SARS-CoV-2 pandemic and the urgent need for massive antiviral testing highlighted the lack of a good cell-based assay that allowed for a fast, automated screening of antivirals in high-throughput content with minimal handling requirements in a BSL-3 environment. The present paper describes the construction of a green fluorescent substrate that, upon cleavage by the SARS-CoV-2 main protease, re-localizes from the cytoplasm in non-infected cells to the nucleus in infected cells. The construction was stably expressed, together with a red fluorescent nuclear marker, in a highly susceptible clone derived from Vero-81 cells. With this fluorescent reporter cell line, named F1G-red, SARS-CoV-2 infection can be scored automatically in living cells by comparing the patterns of green and red fluorescence signals acquired by automated confocal microscopy in a 384-well plate format. We show the F1G-red system is sensitive to several SARS-CoV-2 variants of concern and that it can be used to assess antiviral activities of compounds in dose–response experiments. This high-throughput system will provide a reliable tool for antiviral screening against SARS-CoV-2.

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.

Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to its favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

Study Objective: This study was performed to evaluate the association between uterine weight and operative outcomes in women undergoing laparoscopic hysterectomy for a benign indication.Methods: This is a secondary analysis of a randomized trial with data collected prospectively and retrospectively. The data of 159 women undergoing laparoscopic hysterectomy for a benign indication were analyzed. Women were divided in two groups according to the postoperative uterine weight: small uterus group (

A better understanding of the anti-SARS-CoV-2 immune response is necessary to finely evaluate commercial serological assays but also to predict protection against reinfection and to help the development of vaccines. For this reason, we monitored the anti-SARS-CoV-2 antibody response in infected patients. In order to assess the time of seroconversion, we used 151 samples from 30 COVID-19 inpatients and monitored the detection kinetics of anti-S1, anti-S2, anti-RBD and anti-N antibodies with in-house ELISAs. We observed that specific antibodies were detectable in all inpatients 2 weeks post-symptom onset and that the detection of the SARS-CoV-2 Nucleocapsid and RBD was more sensitive than the detection of the S1 or S2 subunits. Using retroviral particles pseudotyped with the spike of the SARS-CoV-2, we also monitored the presence of neutralizing antibodies in these samples as well as 25 samples from asymptomatic individuals that were shown SARS-CoV-2 seropositive using commercial serological tests. Neutralizing antibodies reached a plateau 2 weeks post-symptom onset and then declined in the majority of inpatients but they were undetectable in 56% of asymptomatic patients. Our results indicate that the SARS-CoV-2 does not induce a prolonged neutralizing antibody response. They also suggest that induction of neutralizing antibodies is not the only strategy to adopt for the development of a vaccine. Finally, they imply that anti-SARS-CoV-2 neutralizing antibodies should be titrated to optimize convalescent plasma therapy.

Objective: The aim of this study was to review the characteristics of patients who underwent laparoscopic removal of giant ovarian cysts using the Alexis Laparoscopic System® and confirm the safety and feasibility of this technique.Method: We conducted a retrospective review of data of women undergoing the procedure from March 2014 to February 2019. Inclusion criteria were ovarian cysts of at least 15 cm. Exclusion criteria were the presence of solid components and suspicion of neoplasia on imaging.Results: Six patients were included in the series. Median size of the cysts at imaging was 22.8 cm (range 15–30 cm), while median volume was 5.9 L (range 1.9–15.6 L). Mean age of operated women was 59 years (range 21–88 years). All patients underwent exclusive laparoscopic management except one patient who underwent a conversion into midline laparotomy. The size of the skin incision initially performed to puncture the cyst ranged from 2.5 to 4 cm. On final pathological reports, two cysts were mucinous cystadenomas, and four were serous cystadenomas. There was no epithelial ovarian cancer or borderline tumor in any of the specimen operated.Conclusion: Laparoscopic management of giant ovarian cysts using the Alexis Laparoscopic System® is safe and feasible in well-selected cases. Midline laparotomy can thus be avoided, decreasing the risk of post-operative complications and increasing quality of life of patients.